Treatment of vascular thrombosis and restenosis with inhaled nitric oxide

ABSTRACT

Disclosed are methods of treating, inhibiting or preventing vascular thrombosis or arterial restenosis in a mammal. The disclosed methods include causing the mammal to inhale a therapeutically-effective concentration of gaseous nitric oxide (NO). Also disclosed are methods that include the administration of the following types of agents in conjunction with inhaled nitric oxide: compounds that potentiate the beneficial effects of inhaled nitric oxide, and antithrombotic agents that complement or supplement the beneficial effects of inhaled nitric oxide.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Work on this invention was supported, in part, with funds from theUnited States government (USPHS grants HL42397 and HL45895). Thegovernment therefore has certain rights in the invention.

This is a division of copending application Ser. No. 08/389,304, filedFeb. 16, 1995.

BACKGROUND OF THE INVENTION

This invention relates to methods of treating, inhibiting or preventingvascular thrombosis and methods of treating, inhibiting or preventingarterial restenosis resulting from excessive intimal hyperplasia.

A thrombosis, i.e, the formation or presence of a blood clot within ablood vessel, may result from physical injury of an arterial wall by avascular interventional procedure such as percutaneous transluminalcoronary angioplasty ("PTCA"; a type of balloon angioplasty) or coronarybypass surgery. Thrombosis may also result from progression of a naturaldisease, such as atherosclerosis. Various agents, including aspirin,prostaglandin E₁, selective thromboxane A₂ inhibitors, selectivethrombin inhibitors, platelet receptor GPIIb/IIIa blockers, tissueplasminogen activator, streptokinase, heparin and kistrin have been usedas antithrombotics. See, e.g., Yasuda et al. (Circulation 83:1038(1991)); and Gold et al., (Circulation (Supplement IV) 83:IV26 (1991)).Currently available antiplatelet agents include aspirin, ticlopidin,monoclonal antibodies, nitroglycerin and sodium nitroprusside.

Another unwanted result of arterial injury from vascular interventionalprocedures is arterial restenosis. For example, PTCA has been usedextensively to open occluded arteries in both the coronary andperipheral vascular systems. Although initially successful in over 95percent of cases, a gradual renarrowing or reocclusion process, known asrestenosis, occurs within six months in 30 to 50 percent of thepatients.

Vascular interventional procedures tend to damage the vascular wall.Such injury triggers the proliferation of vascular smooth muscle cells,resulting in intimal hyperplasia. This proliferation may be due in partto the effects of platelet-derived growth factors released by plateletswhich adhere to the site of arterial injury. Excessive intimalhyperplasia leads to restenosis, i.e., a re-narrowing of the arteriallumen. Thus, arterial restenosis can severely limit the long-termeffectiveness of vascular interventional procedures.

SUMMARY OF THE INVENTION

It has been discovered that inhaled nitric oxide (NO) gas increasesblood vessel patency after lysis of a thrombus at the site of a criticalstenosis, without causing any systemic hemodynamic effects. Theantithrombotic effects of inhaled NO persist after the cessation of NOinhalation. It has also been discovered that inhaled NO inhibitsarterial restenosis resulting from excessive intimal hyperplasia.

Accordingly, in one aspect, the invention features a method fortreating, inhibiting or preventing a thrombosis in a mammal, by causingthe mammal to inhale a therapeutically effective amount of gaseous NO.The thrombosis may be associated with a disease or injury.

In another aspect, the invention features a method for treating,inhibiting or preventing arterial restenosis in a mammal, by causing themammal to inhale a therapeutically effective amount of gaseous NO. Thearterial restenosis may be associated with a disease or injury.

A second compound which potentiates the beneficial effects of inhaled NOmay be administered to a mammal, in conjunction with NO inhalation. Onemeans whereby the second compound could potentiate the beneficialeffects of NO inhalation is by prolongation of the effect of inhaled NOin target tissues, or cells that interact with the target tissues,without causing undesirable systemic vasodilation and concomitantdecrease in systemic blood pressure. More particularly, prolonging theantithrombotic and/or intimal hyperplasia-inhibiting effect of inhaledNO may be accomplished by administering a phosphodiesterase-inhibitingagent in conjunction with (i.e., before, during or immediately after) NOinhalation. Preferred phosphodiesterase inhibitors are those thatselectively inhibit cGMP-specific phosphodiesterases, while minimallyaffecting the breakdown of adenosine 3',5'-cyclic monophosphate (cAMP)by other phosphodiesterases. A preferred phosphodiesterase inhibitordisplaying the aforementioned specificity is Zaprinast™ (M&B 22948;2-o-propoxyphenyl-8-azapurine-6-one, Rhone-Poulenc Rorer, DagenhamEssex, UK). Other cGMP-specific phosphodiesterase inhibitors are knownand may be substituted for Zaprinast™.

Another means whereby the second compound could potentiate thebeneficial effects of NO inhalation is by prolongation of the in vivohalf-life of NO. That may be accomplished, for example, by administeringsuperoxide dismutase. Superoxide dismutase removes superoxide radicals,which react with NO.

In other preferred embodiments, a second antithrombotic agent whichaugments the beneficial antithrombotic effects of inhaled NO may beadministered to a mammal in conjunction with the inhaled NO. Examplesinclude aspirin, ticlopidine, monoclonal antibodies, nitroglycerin, andsodium nitroprusside.

As used herein, "acute ischemic coronary syndrome" means acute occlusionof a coronary artery, e.g., acute myocardial infarction, unstableangina, crescendo angina, angina pectoris, coronary intermediatesyndrome, angina pectoris status post thrombolysis and angina pectorisstatus post PTCA.

As used herein, "antithrombotic agent" means an agent that inhibitsthrombus formation, stimulates thrombolysis, i.e., thrombus dissolution,or both.

As used herein, "intimal (or neointimal) hyperplasia" meansproliferation of arterial smooth muscle cells in the intima, in responseto arterial endothelial denudation.

As used herein, "revascularization" means reestablishment of bloodsupply to a part of the mammalian body.

As used herein, "target cells" means non-pulmonary cells that interactdirectly with NO, following NO inhalation, to mediate a beneficialeffect in the inhibition, prevention or treatment of thrombosis orarterial restenosis.

As used herein, a "therapeutically effective concentration of gaseousNO" is a concentration that induces one or more of the following in agiven patient: relief of chest pain, relief of shortness of breath,resolution of ischemic electrocardiographic changes, and improvedcoronary artery blood flow (which may be shown by angiography).

As used herein, a "therapeutically effective" amount of aphosphodiesterase inhibitor is an amount that increases the duration(i.e., half-time) or magnitude of the therapeutic effect of gaseous NO.

As used herein, "thrombosis" means the formation or presence of a bloodclot within a blood vessel, which clot may cause infarction or ischemiaof tissues supplied by the vessel.

As used herein, "arterial restenosis" means an abnormal narrowing of anarterial lumen as a result of excessive (neo)intimal hyperplasia.

As used herein, "vascular interventional procedure" means any surgicalprocedure that involves an anatomical disruption or a mechanicaldisturbance of a blood vessel. A non-exclusive list of vascularinterventional procedures includes balloon angioplasty, laserangioplasty, coronary artery surgery, atherectomy and coronary arterystents.

The methods herein disclosed are useful for treating, inhibiting orpreventing thrombosis, such as may result from a naturally occurringdisease, e.g., athrosclerosis, or from a vascular interventionalprocedure, e.g., angioplasty or coronary bypass surgery. The methodsherein disclosed are also useful for treating, inhibiting or preventingarterial restenosis resulting from excessive intimal hyperplasia. Sucharterial restenosis is a frequent complication associated with vascularinterventional procedures, such as PTCA or coronary bypass surgery.

According to this invention, NO gas may be administered:

(a) continuously or intermittently;

(b) in the absence of tobacco smoke;

(c) at a concentration between 0.1 and 300 parts per million (ppm),preferably between 1.0 and 200 ppm, and most preferably between 20 and100 ppm;

(d) as a mixture including NO, oxygen (O₂), and nitrogen (N₂) or otherinert gases, preferably having an FiO₂ (i.e., proportion of O₂, byvolume) of 0.20 to 0.99, the proportion of O₂ in air being 0.21, andwhere the NO gas is mixed with the O₂ -containing gas shortly beforeinhalation (e.g., less than 5 minutes before inhalation, and preferablyless than one minute before inhalation); and

(e) in such a way that the concentration of NO₂ is monitored and keptwithin safe limits (e.g., less than 1 ppm).

An important advantage of this invention is that inhaled NO does notinduce systemic vasodilation and concomitant acute systemic hypotension.Systemic vasodilation is an undesirable, potentially dangerous sideeffect associated with sustained systemic NO release from oral orintravenously administered NO donor compounds which have been used toinhibit thrombosis or excessive intimal hyperplasia.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph comparing open arterial time (as a percentage oftotal time) for the control group before, during and after zero ppm NOinhalation, in the canine coronary artery thrombosis model.

FIG. 1B is a graph comparing open arterial time (as a percentage oftotal time) for the 20 ppm inhaled NO group before, during and after NOinhalation, in the canine coronary artery thrombosis model. The asteriskindicates value significantly differs from baseline (p<0.05).

FIG. 1C is a graph comparing open arterial time (as a percentage oftotal time) for the 80 ppm inhaled NO group before, during and after NOinhalation, in the canine coronary artery thrombosis model. The asteriskindicates value significantly differs from baseline (p<0.05).

FIG. 1D is a graph comparing open arterial time (as a percentage oftotal time) for the 200 ppm inhaled NO group before, during and after NOinhalation, in the canine coronary artery thrombosis model.

FIG. 2 is a graph showing ADP-induced canine platelet aggregation curvesfor samples exposed to 0 ppm NO or 400 ppm NO.

FIG. 3 is a graph summarizing dose response data on the effect ofinhaled NO on canine platelet aggregation.

FIG. 4 is a graph comparing the intima/media ratio in control rats; ratsexposed for two weeks to 20 ppm NO; rats exposed for two weeks to 80 ppmNO; and rats exposed for one week to 80 ppm NO, followed by one week ofbreathing air. The asterisk indicates value significantly differs frombaseline (p<0.05).

DETAILED DESCRIPTION

Thrombosis

The invention provides a simple, rapid, selective and efficacious methodfor treating, inhibiting or preventing a vascular thrombosis in amammal, such as a human patient. The thrombosis to be treated, inhibitedor prevented may result from any of the various causes of thromboses.For example, the type of thrombosis may be a posttraumatic arterialthrombosis. The thrombosis may be at any one of various locations in themammalian body. For example, the thrombosis location may be arterial,venous, coronary, cerebral, femoral, renal or placental. Preferably, thelocation of the thrombosis treated, inhibited or prevented is notpulmonary.

Coronary thromboses are often associated with an acute ischemic coronarysyndrome. Any thrombosis-related acute ischemic coronary syndrome may betreated, inhibited or prevented by the administration of inhaled NO,according to this invention. The thrombosis-related acute ischemiccoronary syndrome may be associated with, e.g., an artery-occludingdisease or a vascular interventional procedure. Thrombosis-related acuteischemic coronary syndromes include, but are not limited to, myocardialinfarction, unstable angina pectoris, thrombosis after coronaryrevascularization, and reocclusion after coronary thrombolysis. Coronaryrevascularization may be accomplished by various known vascularinterventional procedures, e.g., PTCA, laser angioplasty, coronaryartery bypass grafting, coronary artery atherectomy or coronary arterystents.

Inhaled NO may be used, according to this invention, to treat a mammalthat has been identified as having an existing thrombosis. Mammals, andhumans in particular, are known to display various signs and symptomsrelating to the existence of a thrombosis, and may be identifiedthereby. The recognition of such signs and symptoms is within the skillof medical practitioners. Signs and symptoms of a thrombosis in a humanpatient include, but are not limited to, the following: chest pain,shortness of breath, paralysis, limb pain, myocardial infarction, anginapectoris, unstable angina pectoris, crescendo angina pectoris, ischemiccongestive heart failure, cardiogenic shock, peripheral vascular diseaseand ischemic limb. In one embodiment of this invention, the identifiedmammal is a human who has a thrombosis, and the thrombosis to be treatedby NO inhalation is manifested as a myocardial infarction.

Alternatively, inhaled NO also may be used to inhibit or preventthrombosis development in a mammal that does not have an existingthrombosis but has been identified as being at risk of developing athrombosis. Certain existing pathological conditions, e.g.,atherosclerosis, are known to put an individual at risk of developing athrombosis. In addition, certain vascular interventional procedures,e.g., balloon angioplasty and coronary artery bypass surgery, are knownto put an individual at risk of developing a thrombosis. Accordingly,individuals diagnosed as having pathological conditions with which anincreased risk of thrombosis is associated, individuals who haveundergone one or more vascular interventional procedures, andindividuals for whom one or more vascular interventional procedures areimminent, may be advantageously treated according to this invention.

Similarly, the identified mammal may be a human at risk of developing athrombosis, where the risk is associated with an artery-occludingdisease such as atherosclerosis. Individuals with such pathologicalconditions may be identified by methods known to medical practitionerstrained in the relevant areas of medical practice.

In another embodiment of this invention, the risk is associated with avascular interventional procedure such as angioplasty. In a particularlypreferred embodiment, the identified mammal is a human at risk ofdeveloping a thrombosis as a result of a PTCA procedure.

Arterial Restenosis

The invention provides a simple, rapid, selective and efficacious methodfor treating, inhibiting or preventing arterial restenosis resultingfrom excessive intimal hyperplasia. Excessive intimal hyperplasia andarterial restenosis frequently occur following vascular interventionalprocedures such as angioplasty of any vessel (e.g., carotid, femoral,coronary, etc.); or any coronary revascularization procedure, includingballoon angioplasty, laser angioplasty, coronary artery bypass grafting,atherectomy or coronary artery stents.

Inhaled NO may be used, according to this invention, to treat a mammalthat has been identified as having an existing arterial restenosis.Mammals are known to display various signs and symptoms relating to theexistence of an arterial restenosis, and may be identified thereby. Therecognition of such signs and symptoms is within the skill of medicalpractitioners. Signs and symptoms of a arterial restenosis in a mammalinclude, but are not limited to, the following: chest pain, shortness ofbreath, electrocardiographic changes and coronary angiographic findings.The objective of treating an existing arterial restenosis with inhaledNO is to reduce the thickness of the intima of a restenosed artery, soas to increase the diameter of the arterial lumen, i.e., reduce thepathological stricture.

Alternatively, inhaled NO may be used, according to this invention, toinhibit or prevent arterial restenosis in a mammal (e.g., a human) thatdoes not have an existing arterial restenosis but has been identified asbeing at risk of developing an arterial restenosis. Certain vascularinterventional procedures, e.g., PTCA and coronary bypass surgery, cancause arterial trauma. Arterial trauma is known to lead to excessiveintimal hyperplasia and arterial restenosis. Therefore, those proceduresput a patient at risk of developing an arterial restenosis. Accordingly,individuals who have undergone one or more vascular interventionalprocedures, and individuals for whom one or more vascular interventionalprocedures are imminent, may be advantageously treated according to thisinvention.

The inhaled NO is, for example, administered to a human patient who isscheduled to undergo a vascular interventional procedure. Inhaled NO maybe administered in advance of a vascular interventional procedure, tominimize the amount of intimal thickening that takes place following thearterial trauma normally associated with vascular interventionalprocedures. A patient receiving inhaled NO as a preventive measurebefore a vascular interventional procedure may or may not have anexisting arterial restenosis. Alternatively, inhaled NO may beadministered to a patient who has already undergone a vascularinterventional procedure. In that situation, administration of theinhaled NO preferably will begin within hours of the vascularinterventional procedure. Of course, one of skill in the art willrecognize that inhaled NO may be administered before, during and after avascular interventional procedure. Regardless of whether the inhaled NOis administered before, during or after the procedure, or all three, theinhaled NO may be administered continuously or intermittently.

Administration of Inhaled NO

Inhaled NO is preferably administered from a source of stored,compressed NO gas. Compressed NO gas may be obtained from a commercialsupplier such as Airco (Murray Hill, N.J.), typically as a mixture of200-800 ppm NO in pure N₂ gas. The source of NO can be 100% NO, ordiluted with N₂ or any other inert gas (e.g., helium). It is vital thatthe NO be obtained and stored as a mixture free of any contaminating O₂or higher oxides of nitrogen, because such higher oxides of nitrogen(which can form by reation of O₂ with NO) are potentially harmful tolung tissues. If desired, purity of the NO may be demonstrated withchemiluminescence analysis, using known methods, prior to administrationto the patient. Chemiluminescence NO-NO_(x) analyzers are commerciallyavailable (e.g., Model 14A, Thermo Environmental Instruments, Franklin,MA). The NO-N₂ mixture may be blended with air or O₂ through, forexample, calibrated rotameters which have been validated previously witha spirometer. The final concentration of NO in the breathing mixture maybe verified with a chemical or chemiluminescence technique well known tothose in the field (e.g., Fontijin et al., Anal. Chem. 42:575 (1970)).Alternatively, NO and NO₂ concentrations may be monitored by means of anelectrochemical analyzer. Any impurities such as NO₂ can be scrubbed byexposure to NaOH solutions, baralyme, or sodalime. As an additionalcontrol, the FiO₂ of the final gas mixture may also be assessed. Ifdesired, the ventilator may have a gas scavenger added to the expiratoryoutlet to ensure that significant amounts of NO will not escape into theadjacent environment.

In a hospital or emergency field situation, administration of NO gascould be accomplished, for example, by attaching a tank of compressed NOgas in N₂, and a second tank of oxygen or an oxygen/N₂ mixture, to aninhaler designed to mix gas from two sources; by controlling the flow ofgas from each source, the concentration of NO inhaled by the patient canbe maintained at an optimal level. NO gas may also be mixed with roomair, using a standard low-flow blender (e.g., Bird Blender, PalmSprings, Calif.). NO may be generated from N₂ and O₂ (i.e., air) byusing an electric NO generator. Such a generator is described in ZapolU.S. patent application 07/850,383 (notice of allowance issued), whichis hereby incorporated by reference. In addition, NO may be providedintermittently from an inhaler. The use of an inhaler may beparticularly advantageous if a phosphodiesterase inhibitor isadministered, orally or by inhalation, in conjunction with the NO.

NO may be administered to a mammal identified as having a thrombosis orarterial restenosis, or a mammal identified as being at risk fordeveloping a thrombosis or arterial restenosis, at a concentration offrom 0.1 ppm to 300 ppm in air, pure oxygen, or another suitable gas orgas mixture, for as long as needed. Preferably, the concentration willbe between 1.0 and 200 ppm; and most preferably between 20 and 100 ppm(e.g., 30 ppm, 40 ppm, 50 ppm, 60 ppm or 80 ppm). The concentration maytemporarily increased for short periods of time: e.g., 5 min at 300 ppmNO, when an immediate dramatic effect is desired. For the reasonsexplained below, concomitant treatment with a phosphodiesteraseinhibitor may decrease the total dosage of NO required (or allowintermittent dosage) to produce a satisfactory anti-thrombotic oranti-restenosis effect.

For treatment, inhibition or prevention of arterial restenosis, it maybe necessary to administer inhaled NO by nasal prongs, mask, tent,intra-tracheal catheter or endotracheal tube, for an extended period,i.e., days or weeks. The administration may be continuous, during theextended period. Alternatively, administration could be intermittentduring the extended period. The administration of gaseous NO may be viaspontaneous or mechanical ventilation.

Assessment of Effects of Inhaled NO

When inhaled NO is administered to treat, inhibit or prevent thrombosisor arterial restenosis, it is desirable to monitor the effects of the NOinhalation. Such monitoring can be used, in a particular individual, toverify desirable effects and to identify undesirable side effects thatmight occur. Such monitoring is also useful in adjusting dose level,duration and frequency of administration of inhaled NO in a givenindividual.

Preferably, the effects of inhaled NO on a patient would be assessed byone or more of the following: clinical manifestations such as chestpain; electrocardiography; serial analyses of vascular patency byultrasound, coronary angiography or other means; and increases in levelsof cGMP in plasma or platelets.

Phosihodiesterase Inhibitors

NO decomposes rapidly by reacting with molecular oxygen to producenitrite and nitrate. In addition, NO entering the blood is rapidlyinactivated by tight binding to hemoglobin. For these reasons, NO hasonly a short half-life in arterial blood. This means that inhaled NOadvantageously avoids systemic vasodilation, an undesirable, potentiallydangerous side effect associated with sustained systemic NO release fromNO donor compounds.

It may be desirable to prolong the beneficial effects of inhaled NOwithin the target cells or within cells interacting with the targetcells in the lung. In the context of thrombosis inhibition, preventionand treatment, circulating platelets are the target cells. In thecontext of arterial restenosis inhibition, prevention and treatment,circulating platelets (and possibly white cells) are the cells whichinteract with the target cells in the vasculature.

In determining how to prolong the beneficial effects of inhaled NO, itis useful to consider that one of NO's in vivo effects is activation ofsoluble guanylate cyclase, which stimulates production ofguanosine-3',5'-cyclic monophosphate (cGMP). At least some of thebeneficial effects of NO appear to result from NO's stimulation of cGMPbiosynthesis. Accordingly, in a preferred embodiment of the invention, aphosphodiesterase inhibitor is administered in conjunction with NOinhalation, to inhibit the breakdown of cGMP by endogenousphosphodiesterases.

The phosphodiesterase inhibitor may be introduced into the mammal by anysuitable method, including via an oral, transmucosal, intravenous,intramuscular, subcutaneous or intraperitoneal route. Alternatively, theinhibitor may be inhaled by the mammal. For inhalation, thephosphodiesterase inhibitor is advantageously formulated as a dry powderor an aerosolized solution having a particle or droplet size of lessthan 10 μm for optimal deposition in the alveoli, and may optionally beinhaled in a gas containing NO.

A preferred phosphodiesterase inhibitor is Zaprinast™ (M&B 22948;2-o-propoxyphenyl-8-azapurine-6-one; Rhone-Poulenc Rorer, DagenhamEssex, UK). Zaprinast™ selectively inhibits the hydrolysis of cGMP withminimal effects on the breakdown of cAMP in vascular smooth muscle cells(Trapani et al., J. Pharmacol. Exp. Ther. 258:269 (1991); Harris et al.,J. Pharmacol. Exp. Ther. 249:394 (1989); Lugnier et al., Biochem.Pharmacol. 35:1743 (1986); Souness et al., Br. J. Pharmacol. 98:725(1989)). When using Zaprinast™ according to this invention, thepreferred routes of administration are intravenous or oral. The suitabledose range may be determined by one of ordinary skill in the art. Astock solution of Zaprinast™ may be prepared in 0.05N NaOH. The stockcan then be diluted with Ringer's lactate solution to the desired finalZaprinast™ concentration, immediately before use.

This invention may be practiced with other phosphodiesterase inhibitors.Various phosphodiesterase inhibitors are known in the art, includingdipyridamole and theophyline. As with Zaprinast™, the route ofadministration and suitable dose range may be determined by one ofordinary skill in the art.

Other Antithrombotic Agents in Conlunction with Inhaled NO

Thrombosis may be treated by agents that inhibit thrombus formation,agents that stimulate thrombolysis, i.e., thrombus dissolution, or both.Examples of antithrombotic agents are aspirin, streptokinase, urokinase,tissue plasminogen activator ("t-PA"), met-t-PA (i.e., t-PA with anN-terminal methionine residue), FE1X (a t-PA analog) heparin, hirudinand Hirulog™ (a hirudin analog). Other antithrombotic agents could alsobe used in the practice of this invention. One or more suchantithrombotic agents may be administered to a mammal before, during orafter treatment with inhaled NO, so that their separate antithromboticactivity is advantageously used to augment the antithrombotic effect(s)of inhaled NO.

For example, in one embodiment of this invention, an appropriate dose oft-PA is administered before, during or immediately after NO inhalation,to treat thrombosis. While the inhaled NO is inhibiting or preventingthe formation of new thrombi, the t-PA will stimulate the dissolutionof: (1) thrombi already present at the time inhaled NO was administered,or (2) thrombi formed (albeit at a reduced rate) during or immediatelyafter the NO inhalation. The selection of appropriate antithromboticagents to be administered in conjunction with inhaled NO, and theselection of the appropriate dosage and route of administration of thoseantithrombotic agents is within ordinary skill in the art.

In a preferred embodiment of this invention, aspirin is administeredbefore, during or immediately after NO inhalation, to treat thrombosis.The preferred aspirin dose range is 81 to 325 mg (orally) per day.

While the two aspects of this invention (i.e., thrombosis and arterialrestenosis) have been described separately, it should be appreciatedthat an individual may already have, or be at risk of developing, both athrombosis and arterial restenosis. Under such circumstances, bothaspects of the invention could be practiced at the same time, in thesame individual, with a single administration of inhaled NO.

EXPERIMENTAL INFORMATION

Thrombosis Experiments

The antiplatelet effects of NO have been assessed in a recognized caninemodel of acute coronary thrombolysis after thrombus formation. Thecanine model used was essentially as described by Yasuda et al. (J. Am.Coll. Cardiol. 13:1409 (1989)) and Gold et al. (Circulation (suppl. IV)83:IV26 (1991)).

Twenty-five adult mongrel dogs (20-25 kg) of either sex wereanesthetized with pentobarbital (30 mg/kg body weight). Supplementalpentobarbital was administered as required to maintain generalanesthesia. The dogs' tracheas were intubated and their lungs weremechanically ventilated at 15 breath/min and 10-15 ml/kg with aventilator at FiO₂ within 21-35% (Hudson Ventronics, Tenecula, Calif.)and adjusted to maintain the arterial blood O₂ between 80 and 100 torr.The oxygen saturation was continuously monitored during the experiment,using a pulse oximeter (Nellcor, Inc., Haywood, Calif.).

The femoral artery and vein were cannulated with a polyvinyl chloridecatheter for continuous arterial pressure, blood sampling and infusion.Lidocaine 0.1 mg/kg/min i.v., was given when arrhythmias occurred.Thoracotomy was performed through the left fifth intercostal space. Thepericardium was opened and suspended to create a pericardial cradle. Theleft anterior descending artery was dissected out free, and a 2.5 cmsegment was isolated distal to the first diagonal branch. One milliliterof blood was withdrawn for thrombus formation. A 0.7 mm i.d. catheterwas inserted into a side branch of the isolated left anterior descendingcoronary artery segment, and an ultrasonic flow probe (T101 TransonicSystem, Inc., Ithaca, N.Y.) was placed on the proximal portion of theartery for continuous blood flow monitoring. A 2 mm wide plastic wire(Mass Gas and Electric Supply, Watertown, Mass.) was progressivelyconstricted around the left anterior descending artery, just distal tothe proposed site of thrombus formation, to limit blood flow to 50±10%of baseline. Previous angiographic study has shown this to decrease theluminal diameter by more than 90%.

The isolated left anterior descending coronary artery was traumatized byfour consecutive external compressions with blunted forceps during 3-5seconds, to damage the endothelium and promote thrombus adherence. Snareocclusions were made distal to the probe and proximal to theconstriction site. Thrombin (0.1 ml of 100 units/ml; Thrombinar, ArmourPharmaceutical, Kankakee, Ill.) mixed with 0.3 ml of blood was injectedthrough the side branch catheter into the emptied coronary arterysegment to induce thrombus formation. After 10 minutes, the proximalsnare was released. Two minutes later, the distal snare was released.Ten minutes after thrombus formation, a heparin bolus (75 UI/kg) wasadministered intravenously and followed by a continuous heparin infusion(50 UI/kg/h). After a 30-minute period of stable occlusion, recombinantt-PA boluses (0.45 mg/kg; Activase™, Genentech Inc., South SanFrancisco, Calif.) were administered at 15-minute intervals, untilrecanalization of the thrombosed coronary artery was achieved or amaximum of four boluses had been administered.

This procedure induced alternating periods of recanalization andreocclusion (defined as less than 25% of poststenotic flow) afterinitial reflow of the left descending coronary artery. The ratio openingtime/total time was recorded as the primary outcome variable. ECG,systemic arterial pressure, left atrial pressure and coronary blood flowwere continuously recorded. Animals with: (1) no reperfusion, (2)reperfusion without occlusion, (3) fewer than 3 cycles during the first45-minute observation period, or (4) death before the end of the firstobservation period, were excluded from further study.

NO gas (800 ppm NO in nitrogen, Airco, Murray Hill, N.J.) was mixed withroom air using a standard low-flow blender (Bird Blender, Palm Springs,Calif.) and then titrated with varying quantities of NO, to maintain aconstant FiO₂ just prior to delivery to the ventilator. Inspired NOlevel was continuously monitored by a chemiluminescence NO-NO_(x)analyzer (Model 14A, Thermo Environmental Instruments, Franklin, Mass.;Fontijin et al., supra). The FiO₂ was measured (oxygen meter No. 5590,Hudson, Temecula, Calif.) distal to the reservoir bag after theNO-containing gases were mixed. The exhaled gases, as well as thosedischarged from the chemiluminescence analyzer, were scavenged by use ofa venturi exhalation trap maintained at negative atmospheric pressuredby the laboratory's central vacuum system. The ambient NO/NO₂ levels, asmeasured intermittently by chemiluminescence, did not increase duringthe experiments. At each time samples were taken and methemoglobinlevels were measured. Heart rate, blood pressure and coronary flow weremonitored and recorded continuously.

After a 45 minute baseline study period (pretreatment period) theanimals were divided into four groups: Group A (n=6) was given 0 ppminhaled NO (i.e., air) for 45 minutes; Group B (n=6) was given 20 ppminhaled NO for 45 minutes; Group C (n=6) was given 80 ppm inhaled NO for45 minutes and Group D (n=6) was given 200 ppm for 45 minutes. Allgroups of animals were then observed for a third study period beginningwith the cessation of NO administration and lasting 45 minutes(posttreatment period).

In vitro Measurement of Platelet Aarrecation

Blood from 8 dogs was collected in 0.01M citrate and centrifuged at roomtemperature at 370×g for 5 minutes, for the preparation of platelet-richplasma (PRP), and at 1200×g for 10 minutes, for the preparation ofplateletpoor plasma (PPP). PRP was exposed only to plastic containersor, during testing, siliconized glassware. The platelet count(Thrombocounter C™ platelet counter, Coulter Electronics, Inc., Hialeah,Fla. of PRP was adjusted by dilution with PPP to obtain 300,000/mm³±10%. PRP was then aliquoted into cuvettes incubated at 37° C. withmagnetic stirring (1000 rpm) in a dual channel aggregometer (Model 440,Chrono-Log Corp., Havertown, Pa.). Light transmission was continuouslyrecorded on a recorder (Model 707, Chrono-Log Corp., Havertown, Pa.).The aggregation sample tube was obstructed with a rubber cap and two 19gauge needles (Sherwood Medical, St. Louis, Mo.) were placed throughthis cap, allowing the delivery of a mixture of NO gas and oxygen abovethe PRP. The inlet needle was connected via a flowmeter to a gasreservoir into which a mixture of NO gas, air and oxygen titrated asdescribed above was delivered. The NO concentration was continuouslymonitored by the chemiluminescence NO-NO_(x) analyzer. A needle valveallowed regulation of the flow through the sample to approximately 40ml/min. Positive pressure was maintained in the system by having theoutlet needle connected to a recipient flask filled with 5 cm of waterin which constant bubbling was maintained.

ADP-induced platelet aggregometry studies were performed after theexposure of the test cuvette containing 450 μl of adjusted PRP to the NOgas mixture for 10 minutes. Gas administration was continued during themeasurement of ADP-induced aggregation. Platelet aggregation was studiedusing different aliquots of PRP treated with 20, 80, 200 and 400 ppm ofNO, in random order. Control ADP-induced platelet aggregation studieswithout NO were performed before and after the administration of NO toassess the stability of the PRP preparation. All experiments werecompleted within 4 hours of blood collection.

Data Analysis

Artery patency was defined as the fraction of the total observationperiod during which flow was greater than 25% of the basic flow afterthe initial stenosis was created. Except as noted, results are expressedas mean±SEM. The significance of differences between groups wasdetermined with Student's t-test for paired or unpaired values asappropriate. The significance of the dose response of the effect of NOon ADP-induced platelet aggregation was assessed with two-way analysisof variance. A p value <0.05 was considered significant.

Canine Thrombolysis and Reperfusion Model Results

In each experimental group, the external constrictor reduced leftanterior descending artery blood flow by 54±2% of baseline, from 22±2 to10±1 ml/min. The median number of tPA boluses required to obtainreperfusion was two, with a range of 1 to 4 (Table 1). Cyclic reflow andreocclusion accompanied by electrocardiographic evidence of myocardialinjury occurred in all animals except one in which reocclusion did notoccur; this animal was excluded from further study.

                                      TABLE 1    __________________________________________________________________________    Baseline Values of Physiological Parameters               Group A                      Group B                             Group C                                    Group D               0 ppm NO                      20 ppm NO                             80 ppm NO                                    200 ppm NO    Parameter Measured               (n = 6)                      (n = 6)                             (n = 6)                                    (n = 6)    __________________________________________________________________________    Post-stenotic flow               51.7 ± 3.5                      55.2 ± 3.7                             51.6 ± 4.7                                    54.6 ± 1.9    (% before stenosis)    Number of tPA boluses                2.0 ± 0.4                       1.7 ± 0.2                              1.8 ± 0.3                                     2.0 ± 0.4    ACT (sec)  219 ± 16                      219 ± 14                              236 ± 5.3                                     196 ± 12.8    FIO.sub.2 (%)               29.0 ± 1.1                      27.5 ± 1.0                             32.2 ± 1.5                                    31.6 ± 3.0    Ratio (r)  55.7 ± 8.5                      50.6 ± 6.9                             48.8 ± 2.9                                    45.2 ± 5.6    Hb (mg/dl) 13.26 ± 1.35                      14.28 ± 0.85                             14.08 ± 0.85                                    12.95 ± 0.64    Platelet Ct (× 10.sup.3 /mm.sup.3)               301 ± 55                      292 ± 25                             277 ± 35                                    237 ± 35    MetHb (%)   0.25 ± 0.17                       0.10 ± 0.06                              0.18 ± 0.09                                     0.26 ± 0.09    __________________________________________________________________________

Table 1: "Number of tPA boluses" refers to number of boluses injected;"ACT" refers to activated clotting time; "FIO₂ " refers to inspired O₂fraction; "Ratio" refers to unoccluded/occluded coronary artery atbaseline; "Platelet Ct" refers to platelet count; and "MetHb" refers tomethemoglobin (percentage of total hemoglobin). There is no significantdifference between the baseline flow rate ratios at 0 ppm (control), 20ppm, 80 ppm and 200 ppm of inhaled NO (n=6 for each group).

Effects of Inhaled NO on Artery Patency

In animals receiving 0 ppm inhaled NO (Group A), arterial patency didnot change during the three treatment periods (FIG. 1A). In animalsreceiving 20 ppm inhaled NO (Group B), arterial patency increased from50.6±6.9% during the pre-inhalation baseline period, to 63.8±7.90 duringthe inhalation period (p<0.01) (FIG. 1B). In animals receiving 80 ppminhaled NO (Group C), arterial patency increased from 48.8±2.9% duringthe pre-inhalation baseline period, to 75.1±6.7% during the inhalationperiod (p<0.01) (FIG. 1C). In animals receiving 200 ppm inhaled NO(Group D), arterial patency increased from 45.27±5.6% during thepre-inhalation baseline period to 54.8±10.4% during the inhalationperiod (p=NS) (FIG. 1D). In group C the increased arterial patencyobserved during the NO inhalation period persisted during the 45-minutepostinhalation period (70.1±7.1% vs. baseline, p<0.05).

When the inhalation period arterial patency results from groups B, C,and D were pooled, there was a statistically significant differencebetween the pooled treatment groups and the control group (p<0.005).When the 45-minute post-inhalation period artery patency results fromgroups B, C, and D were pooled, there was also a statisticallysignificant difference between the pooled treatment groups and thecontrol group (p<0.005).

Neither platelet count nor blood hemoglobin changed during or after NOadministration in any of the groups of animals (Table 3). Methemoglobinlevels increased from 0.2±0.1% to 1.1±0.4% in the dogs breathing 200 ppminhaled NO (Table 3). This increase persisted after the NOadministration was stopped and was not seen in dogs receiving lowerdoses of NO.

Systemic arterial pressure decreased in all groups of dogs during thisstudy, and did not differ between the dogs receiving NO and those whodid not (Table 2A). Left atrial pressure was unchanged in all groups ofanimals throughout the duration of the study (Table 2B).

                  TABLE 2A    ______________________________________    Mean Systemic Arterial Pressure (SAP) (mmHG)    Before, During and After NO Inhalation    NO Treatment    Before     During  After    ______________________________________    0 ppm NO (N = 6) Group                    89 ± 8  82 ± 6                                       80 ± 7    20 ppm NO (n = 6) Group                    105 ± 6 96 ± 6                                       82 ± 6    B    80 ppm NO (n = 6) Group                    91 ± 5  81 ± 9                                       78 ± 7    C    200 ppm NO (n = 6)                    94 ± 3  95 ± 4                                       93 ± 4    Group D    ______________________________________     Values are expressed as mean ± SEM.

                  TABLE 2B    ______________________________________    Mean Left Atrium Pressure (LAP) (mmHG)    Before, During and After NO Inhalation    NO Treatment   Before    During    After    ______________________________________    0 ppm NO (N = 6) Group                   5.5 ± 0.6                             5.4 ± 0.7                                       5.4 ± 0.7    20 ppm NO (n = 6) Group                   5.2 ± 1.6                             4.7 ± 1.4                                       4.6 ± 1.1    B    80 ppm NO (n = 6) Group                   5.3 ± 0.6                             5.2 ± 0.6                                       4.9 ± 0.6    C    200 ppm NO (n = 6)                   4.6 ± 1.1                             4.2 ± 0.9                                       3.7 ± 0.8    Group D    ______________________________________     Values are expressed as mean ± SEM.

Effects of NO on ADP-Induced In Vitro Platelet Aacrecation

Addition of 20, 80, 200 and 400 ppm NO to the gas mixture above the PRPled to a dose-related decrease in the maximal change in lighttransmission caused by ADP (FIG. 3). There was no change in theADP-induced decrease in light transmission in the two controlaggregation curves performed at the beginning and end of each study(30.57±5.57% vs. 31.1±4.31%).

                  TABLE 3    ______________________________________    Hematologic Values Before, During and After NO Inhalation    NO    Treatment            Measurement                       Before    During  After    ______________________________________    0 ppm NO            Hb (mg/dl) 13.2 ± 1.3                                 13.7 ± 0.8                                         12.2 ± 0.7    Group A Platelet Count                       301 ± 55                                 284 ± 53                                         276 ± 35    (n = 6) (× 10.sup.3 /mm.sup.3)            MetHb (%)  0.22 ± 0.2                                  0.2 ± 0.1                                          0.2 ± 0.2    20 ppm NO            Hb (mg/dl)  14.3 ± 0.85                                 12.7 ± 0.7                                          2 ± 1    Group B Platelet Count                       292 ± 25                                 269 ± 22                                         253 ± 16    (n = 6) (× 10.sup.3 /mm.sup.3)            MetHb (%)   0.1 ± 0.1                                  0.4 ± 0.2                                          0.1 ± 0.02    80 ppm NO            Hb (mg/dl) 14.0 ± 0.8                                 11.9 ± 0.3                                         11.1 ± 0.6    Group C Platelet Count                       277 ± 35                                 238 ± 32                                         209 ± 37    (n = 6) (× 10.sup.3 /mm.sup.3)            MetHb (%)   0.2 ± 0.1                                  0.4 ± 0.1                                          0.3 ± 0.1    200 ppm NO            Hb (mg/dl) 12.9 ± 0.6                                 12.2 ± 1.0                                         12.9 ± 0.9    Group D Platelet Count                       237 ± 35                                 232 ± 44                                         244 ± 40    (n = 6) (× 10.sup.3 /mm.sup.3)            MetHb (%)   0.2 ± 0.1                                  1.1 ± 0.4*                                          0.9 ± 0.3    ______________________________________     Values are expressed as mean ± SEM.     * < 0.05 vs control value.

The above results demonstrate that inhaled NO increases coronary arterypatency after lysis of a thrombus at the site of a critical stenosiswithout producing any systemic hemodynamic effects. They also show thatthe antithrombotic effects of inhaled NO persist for at least 45 minutesafter cessation of NO inhalation. At NO concentrations similar to thoseobtained in vivo, gaseous NO markedly inhibited ADP-induced plateletaggregation in vitro.

Intimal Hyperplasia Experiments

Neointimal smooth muscle cell hyperplasia is the main pathologic processin human arterial restenosis. Therefore, to elucidate the effect ofinhaled NO on the process of restenosis after a vascular interventionalprocedure such as PTCA, the effect of inhaled NO on intimal hyperplasiawas studied, using a rat carotid artery model of arterial injury (Cloweset al., Laboratory Invest. 49:327 (1983)), a standard animal model forhuman neointimal smooth muscle cell hyperplasia.

Arterial Injury

Adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington,Mass.) underwent balloon injury of the common carotid artery in a mannerpreviously shown to bring about neointimal hyperplasia (Clowes et al.,supra). Rats weighing 300 to 350 g were anesthetized by intraperitonealinjection of ketamine (60-80 mg/kg) and acepromazine (0.1 mg/kg). Oncesatisfactory anesthesia had been achieved, a longitudinal midlineincision was made and the left carotid artery was isolated via bluntdissection. After further careful dissection of the internal andexternal carotid bifurcation, the distal external carotid segment wasligated with a 4-0 silk suture. A small arteriotomy was made in theexternal carotid using microdissecting scissors and a 2 French Fogartyballoon catheter (Baxter Edwards LIS) was inserted through thearteriotomy and advanced approximately two centimeters below the carotidbifurcation. The balloon was filled with enough saline to cause visibledistention of the common carotid artery and gently withdrawn to thelevel of the bifurcation. The balloon catheter was then withdrawn andafter allowing back bleeding through the arteriotomy site to eliminatepotential thrombus and air bubbles, the external carotid was ligatedproximal to the arteriotomy site using 4-0 silk sutures. After visualinspection to insure adequate pulsation of the common carotid artery,the surgical lesion was closed and the animals were allowed to recoverfrom anesthesia.

Chronic NO Inhalation

Chronic NO inhalation was carried out in specially prepared 40 literacrylic inhalation chambers. The gas mixtures were blended usingseparately regulated and calibrated flow meters for oxygen, pressurizedair and NO stock gases (800 ppm and 10,000 ppm NO in N₂, Airco, MurrayHill, N.J.). The effluent gas from the chamber was analyzed periodicallythroughout the experimental period, to ensure stable FIO₂ and NO levels.The FIO₂ was measured using a polarographic electrode (Hudson OxygenMeter 5590, Temecula, Calif.), NO concentration was measured bychemiluminescence (Model 14A, Thermo Environmental Instruments, Inc.,Franklin, Mass.); and nitrogen dioxide and nitrogen with higheroxidation states (NO_(x)) were measured by chemiluminescence followingconversion of the NO_(x) to NO by a heated stainless steel oven (850°C.) with 98% efficiency (Model 100B NO_(x) generator, ThermoEnvironmental Instruments, Inc., Franklin, Mass.). Fresh soda lime wasmaintained in the chambers to reduce NO_(x). NO concentration wasmaintained at 20 to 80 ppm, depending upon the experiment. Serialmeasurements revealed FI0₂ to be 21% and NO_(x) to be 3-4 ppm. The gasesexiting the exposure chambers, as well as those discharging from thechemiluminescence instrument, were scavenged using a venturi trapmaintained at negative atmospheric pressure with reference to thelaboratory's central vacuum system.

Control animals were maintained in filtered cages in the same room asthe NO-treated animals. The NO exposure was begun 30 to 120 minutesprior to the surgical procedure. During the surgical procedure, theNO-treated group was taken out of the chamber and exposed to NO by amodified face mask fed from the inflow gas tubing of the chambers.Inhaled gas analysis revealed NO levels of 50-80 ppm during the 80 ppmexperiments and 10-20 ppm during the 20 ppm surgical experiments. Thetotal time each animal was out of the chamber and under the face maskranged from 20 to 40 minutes.

Morphometric Analysis

At 1, 3 and 14 days after balloon injury, the rats were euthanized bylethal intraperitoneal injection of sodium pentobarbital or ketamine. A16 gauge catheter was introduced into the ascending aorta via the leftventricular apex. The descending thoracic aorta was ligated. The animalwas then perfused at a pressure of 100 mm Hg with 100 ml of normalsaline followed by 50 ml of 2% paraformaldehyde in phosphate bufferedsaline (PBS). After in vivo fixation for 15 to 30 minutes, both commoncarotid arteries were isolated and underwent further overnight fixationby immersion in 1% paraformaldehyde in PBS. The tissue was dehydratedusing sequentially increasing concentrations of ethanol followed byxylene, and then embedded in paraffin. Cross sections (6 μm) were cutand stained with hematoxylin and eosin and/or elastin for analysis. Asingle section 7 to 8 mm proximal to the carotid bifurcation was usedfor analysis in each animal. The section being analyzed was photographedat 100×. The image was digitized using a Kodak 2135™ scanner. Theintimal and medial area analyses were performed on a Power Macintosh8100/80™ computer using the public domain NIH Image program (written byWayne Rasband at the U.S. National Institutes Of Health and availablefrom the Internet by anonymous ftp from zippy.nimh.nih.gov or on floppydisk from NTIS, 5285 Port Royal Rd., Springfield, Va. 22161; part numberPB93-504868). The morphometric analysis was performed with theinvestigator blinded as to the experimental group.

Statistical Analysis

Statistical analysis was carried out utilizing commercially availableStatviewr™ (Abacus Concepts) software for Macintosh™ computers. Theexperimental group was compared with the control group using an unpairedtwo tailed t-test. A value of P<0.05 was considered statisticallysignificant. All data are represented as mean+/-SEM. The n value refersto the number of animals per group.

Results from Rodent Model of Restenosis

A total of 62 rats underwent the carotid injury procedure. Five of therats died due to vascular complications during the arteriotomy or theballoon injury, giving a procedure mortality rate of 8%. There was nostatistically significant maldistribution of the deaths among thedifferent experimental groups. There were no post-procedure deaths amongthe 57 rats who survived the initial procedure. All rats, regardless oftheir experimental group, exhibited normal grooming behavior andactivity levels throughout the study. Other than occasional ptosis onthe side of the carotid injury, no rats exhibited a gross neurologicaldeficit. Analysis of a subset of the experimental groups revealed weightgain without statistical difference during exposure to air, NO at 20 ppmand NO at 80 ppm.

In the first series of experiments, the rats undergoing carotid ballooninjury were exposed to either ambient air (n=12) or 80 ppm NO (n=13) inair, throughout the duration of the study. The animals were sacrificed14 days after injury for morphometric analysis of the injured carotidartery. Examination by light microscopy revealed that three rats fromthe air (control) group and one rat from the NO-treated group hadthrombotic occlusion of the lumen of the injured carotid. These animalswere excluded from further analysis. As expected, blood vessels fromboth groups exhibited a loss of endothelium and the development ofneointimal hyperplasia. Examination by light microscopy did not reveal aqualitative difference in the cellular morphology of the neointima (datanot shown). Quantitive analysis, however, revealed that the 80 ppminhaled NO treatment resulted in a 38% inhibition of neointimalhyperplasia, i.e., intima/media ratio of 0.932+/-0.13 in 80 ppm animals,compared to 1.512+/-0.147 in 0 ppm NO (control) animals (P=0.008) (FIG.4).

In the next series of experiments, the effect of a one week exposure toNO was tested. The experimental group (n=7) was exposed to 80 ppm NO forseven days and then transferred to air for an additional seven days,prior to sacrifice. The control group (n=5) was exposed to air for theentire 14 day period. There was no qualitative morphologic differencebetween the groups. In addition, quantitive analysis of the intima/mediaratio revealed no significant difference in neointimal hyperplasiabetween the two groups, i.e., 1.059+/-0.239 in NO-treated animals,compared to 1.217+/-0.309 in controls (P=not significant) (FIG. 4).

In a third series of experiments, a lower inhaled dose of NO wasstudied: the rats breathed either 20 ppm NO (n=8) or air (n=8). Again,no quantitative or qualitive difference in the degree of neointimalhyperplasia could be found between the 2 groups, i.e., intima/mediaratio of 0.917+/-0.233 for 20 ppm NO-treated animals, compared to0.985+/-0.149 for air-breathing controls (P=not significant) (FIG. 4).

The above results demonstrate that inhalation of 80 ppm NO, over atwo-week period, significantly inhibits neointimal hyperplasia followingballoon-induced injury to carotid arteries, in a rodent model.

Other embodiments of the invention are within the following claims.

What is claimed is:
 1. A method for treating, inhibiting or preventingvascular thrombosis in a mammal, which method comprises the steps of:(a)identifying a mammal that has an existing thrombosis or is at risk fordeveloping a thrombosis; and (b) causing said mammal to inhale atherapeutically effective concentration of gaseous nitric oxide (NO);and (c) administering to said mammal a therapeutically effective amountof a phosphodiesterase inhibitor without inducing acute systemichypotension.
 2. The method of claim 1, wherein said mammal is a human.3. The method of claim 1, wherein said thrombosis is an arterialthrombosis.
 4. The method of claim 1, wherein said thrombosis is avenous thrombosis.
 5. The method of claim 1, wherein said thrombosis isnot a pulmonary thrombosis.
 6. The method of claim 1, wherein saidmammal has or is at risk of developing an acute ischemic coronarysyndrome.
 7. The method of claim 6, wherein said acute ischemic coronarysyndrome is selected from the group consisting of myocardial infarction,unstable angina pectoris, thrombosis after coronary revascularization,and reocclusion after coronary thrombolysis.
 8. The method of claim 6,wherein said acute ischemic coronary syndrome is associated with anartery-occluding disease.
 9. The method of claim 6, wherein the acuteischemic coronary syndrome is associated with a vascular interventionalprocedure.
 10. The method of claim 9, wherein said vascularinterventional procedure is selected from the group consisting ofangioplasty, coronary artery surgery and coronary artery stents.
 11. Themethod of claim 10, wherein the angioplasty is percutaneous transluminalcoronary angioplasty (PTCA).
 12. The method of claim 1, wherein said NOis inhaled in a predetermined concentration range.
 13. The method ofclaim 12, wherein said concentration range is 0.1 ppm to 300 ppm. 14.The method of claim 12, wherein said concentration range is 1.0 ppm to200 ppm.
 15. The method of claim 12, wherein said concentration range is20 ppm to 100 ppm.
 16. The method of claim 1, wherein said gaseous NO isinhaled as a mixture comprising NO, oxygen (O₂), and nitrogen (N₂)gases.
 17. The method of claim 16, wherein said mixture comprisesbetween 20% and 99% O₂ gas, by volume.
 18. The method of claim 16,wherein said O₂ is mixed with said NO immediately before said mixture isinhaled by the mammal.
 19. The method of claim 1, wherein saidphosphodiesterase inhibitor is selected from the group consisting of2-o-propoxyphenyl-8-azapurin-6-one, dipyridamole and theophyline. 20.The method of claim 1, further comprising administering to said mammal,in conjunction with NO inhalation, a therapeutically effective amount ofa second antithrombotic agent.
 21. The method of claim 20, wherein saidsecond antithrombotic agent is selected from the group consisting ofaspirin, ticlopidine, streptokinase, urokinase, tissue plasminogenactivator (t-PA), met-t-PA, FE1X, heparin, hirudin and a hirudin analog.22. The method of claim 1, wherein said gaseous NO is inhaled in theabsence of tobacco smoke.
 23. The method of claim 1, wherein said NO isinhaled continuously for an extended period.
 24. The method of claim 1,wherein said NO is inhaled intermittently for an extended period.